Characterisation of putrescine uptake by cultured adult mouse hepatocytes.
Uptake of polyamines by cultured cells has been shown to be influenced by growth rate and/or differentiation. In this study, we have investigated whether the fully differentiated, non-proliferating adult mouse hepatocyte is capable of accumulating extracellular putrescine. When hepatocytes were cultured from 4 to 48 h, uptake of putrescine was found to increase substantially with time spent in culture. The Vmax for putrescine uptake increased 22-fold during this period with no change in apparent Km. Treatment of the cells with cycloheximide or actinomycin D at concentrations that did not affect cell viability inhibited the induction of putrescine uptake. Endogenous putrescine levels increased from 19.7 nmol/mg DNA after 4 h in culture to over 500 nmol/mg DNA after 48 h in culture. This increase was accompanied by a loss of over 90% of ornithine decarboxylase activity. Spermidine levels did not change over this time period, whereas spermine levels decreased by 35%. Difluoromethylornithine prevented the observed increase in intracellular putrescine but did not affect putrescine uptake. The increase in putrescine transport was not inhibited by culturing the hepatocytes in a high concentration of putrescine, spermidine or spermine. Moreover, the induction process was not stimulated by foetal calf serum but was selectively inhibited by the differentiating agents dimethylsulfoxide and retinoic acid. The results from those studies show that cultured mouse hepatocytes express a putrescine transport system that is poorly regulated by extracellular polyamines. The expression of the transporter requires the synthesis of mRNA and protein, and appears to be related to a time-dependent change in hepatocyte phenotype.
Martin
R L
RL
Department of Pharmacology, University of Western Australia, Nedlands.
Ilett
K F
KF
Minchin
R F
RF
D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009955:Ornithine Decarboxylase / Q000378:metabolism; D011073:Polyamines / Q000032:analysis; D011700:Putrescine / Q000378:metabolism*
D002087:Butyrates; D011073:Polyamines; D020148:Butyric Acid; D003609:Dactinomycin; D003513:Cycloheximide; D009955:Ornithine Decarboxylase; D011700:Putrescine; D004121:Dimethyl Sulfoxide